Paraguay is a key spot in the central region of South America where several ecoregions converge. Its fauna (and specifically its herpetofauna) is getting better studied than years before, but still there is a lack of information regarding molecular genetics, and barcoding analyses have proven to be an excellent tool in this matter. Here, we present results of a barcoding analysis based on 16S rRNA gene sequences, providing valuable data for the scientific community in the region. We based our fieldwork in several areas of Paraguay. We analyzed 249 samples (142 sequenced by us) with a final alignment of 615 bp length. We identified some taxonomic incongruences that can be addressed based on our results. Furthermore, we identify groups, where collecting efforts and research activities should be reinforced. Even though we have some blanks in the geographical coverage of our analysis—and there is still a lot to do towards a better understanding of the taxonomy of the Paraguayan herpetofauna—here, we present the largest genetic dataset for the mitochondrial DNA gene 16S of reptiles (particularly, Squamata) from Paraguay, which can be used to solve taxonomic problems in the region.
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